Goblet cells (GCs) in the conjunctiva are specialized epithelial cells producing mucins on the ocular surface. GCs play important roles in maintaining homeostasis of the ocular surface, and GC dysfunction is associated with various complications including dry eye diseases. Current GC examination methods, which are conjunctival impression cytology and confocal reflection microscopy, have limitations for routine examination. Fluorescence microscopy using moxifloxacin was recently introduced as a non-invasive and high-contrast imaging method, but further development is needed to be used for GC examination. Here we developed a non-invasive high-speed high-contrast GC imaging method, called moxifloxacin based axially swept wide-field fluorescence microscopy (MBAS-WFFM). This method acquired multiple fluorescence images with the axial sweeping of the focal plane to capture moxifloxacin labeled GCs on the tilted conjunctival surface in focus and generated all-in-focus images by combining the acquired images. The imaging field of view and imaging speed were increased to 1.6 mm × 1.6 mm and 30 fps. An image processing method was developed for the analysis of GC density. MBAS-WFFM was applied to alkali burn mouse models and detected GC damage and recovery via longitudinal imaging. MBAS-WFFM could assess the status of GCs rapidly and non-invasively. We anticipate MBAS-WFFM to be a starting point for non-invasive GC examination and the diagnosis of GC associated diseases. For example, MBAS-WFFM could be used to classify dry eye diseases into detail categories for effective treatment.
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